Project Summary Alzheimer?s disease (AD) is the most common form of dementia among older people with no cure or effective treatment. A thorough understanding of its molecular mechanisms is required for discovering novel diagnostic and therapeutic strategies against AD. Chemical modifications of DNA such as methylation play critical roles in regulating gene expression and many other key biological processes, and altered DNA methylation pattern has been implicated in brain aging and AD. While much attention has focused on DNA methylation at the fifth position on cytosine (5mC), recent research identified a new form of DNA modification at the sixth position on adenine (6mA) in mammalian brains. However, little is known about its presence, genomic distribution, and possible functions in human brain and relevance to AD. Our preliminary data in mouse and human brain indicated that 6mA is dynamically responsive to environmental stress and accumulates in human AD brain. Our central hypothesis is that altered signature of 6mA modification is causally associated with AD neuropathology. The objectives of this project are to generate the first detailed map of brain 6mA methylome and identify causative genes harboring aberrant 6mA alterations associated with quantitative neuropathological measures for early features of AD pathology (e.g., amyloid plaques, neurofibrillary tangles). To achieve this, we propose three specific aims: (1) Genome-wide mapping of brain DNA 6mA methylome to identify differentially methylated genes/regions harboring altered 6mA sites (D6AMRs) associated with AD pathology in 1,200 postmortem brain tissue samples collected by two large, community-based population cohorts of aging and dementia. (2) Integrated multiomics analysis to elucidate the potential mechanistic role of 6mA alteration in AD pathology; and (3) Functionally validation of top-ranked candidate genes in 3D brain organoids derived from human iPSCs. This innovative project leverages the wealth of deep clinical and neuropathological phenotypes along with rich omics data including genetic (GWAS, WGS), epigenetic (5mC, 5hmC, 6mA, H3K9Ac), and transcriptome (RNA-seq) profiled on the same prefrontal cortex, and will provide unprecedented opportunities to uncover novel molecular mechanisms implicated in AD pathology. Our proposal brings together an exceptionally strong and unique multidisciplinary team with complementary expertise in genetic epidemiology, statistical genetics, bioinformatics, molecular and neuroepigenetics, and Alzheimer?s research. The work proposed represents the frontier in the interface between AD and omics research. Findings of this study will provide novel mechanistic insight into AD pathogenesis, and are likely to discover new molecular targets with important clinical and translational implications.